The present invention, in some embodiments thereof, relates to flow cytometry, and more particularly, but not exclusively, to fluorescene-activated cell sorting (FACS).
Flow cytometry is a technique for simultaneous multiparametric analysis of physical and/or chemical characteristics of single cells. Flow cytometry allows for counting, examining, and sorting cells (or other microscopic particles) and provide multiparametric analysis of the physical and/or chemical characteristics of single particles. The cells to be analyzed flow towards an inspection point, and this flowing is designed such that the particles arrive at the inspection point single file, so each particle is analyzed individually.
One particular flow cytometry technique is termed Fluorescence-Activated Cell Sorting (FACS). FACS provides sorting a heterogeneous mixture of biological cells or other particles into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
The technology has applications in a number of fields, including molecular biology, pathology, immunology, plant biology and marine biology.
For instance, in the field of cell biology and immunology it is used with fluorescence tagged antibodies, which bind to specific target cells and allow quantitative analysis of the antigens in the cytometer. This method is widely used to study protein expression and localization, cell surface antigens quantification (CD markers on blood cells in different clinical pathologies, for instance, leukemia), intracelluar and nuclear antigens (activation of transcription factors such as FoxP3 in T-regulatory cells), cell viability (quantification of apoptotic cells by Annexin/PI staining), and others.
Flow cytometry and FACS also have broad application in medicine, especially in transplantation, hematology, tumor immunology and chemotherapy, genetics and sperm sorting in IVF.
Generally, in a flow cytometer a beam of light is directed onto a stream of fluid, where the particles are aligned in the fluid one by one. A number of detectors are aimed at the point where the stream passes through the light beam. Each suspended particle passing through the beam interacts with the light in some way (for instance, scatters the light), and the light from the particle is picked up by the detectors. The detected light is analyzed to provide various types of information about the physical and chemical structure of each individual particle.
A flow cytometer typically has 5 main components:
a flow cell, where liquid stream carries and aligns the cells so that they pass single file through the light beam for sensing;
a light source;
a detector
an amplification system; and
a computer for analysis of the amplified detected signals.
Aligning the particles in the flow cell is achieved by applying to the particles an external force. One method of applying external force that is used in commercially available flow cytometers is termed in the art “sheath flow”. Other methods include applying to the particles light and ultrasound.